We propose to study the mechanisms that control distinct phases of the development of cortical layer 5 and layer 6 efferent projections to the brainstem and spinal cord, including their pathfinding and the molecular control of their recognition and innervation of targets. We will test the hypothesis that the pioneering of the major path in and out of the cortex by cortical subplate axons through this path. This will be done by creating transgenic mice engineered to have stunted subplate axons, or lack subplate neurons altogether, and by analyzing Mash-1 mutant mice that fail to form a thalamocortical projection. To test the idea that the axonal chemoattractant, Netrin-1, molecularly defines the subcortical path of layer 5 corticospinal axons through the forebrain, midbrain and hindbrain, we will correlate this axonal path with the pattern of Netrin-1 expression, and will analyze mutant mice deficient for Netrin-1 or its receptor, DCC, for aberrant pathfinding by corticospinal axons. The remaining aims focus on target recognition by corticospinal axons, which is characterized by a de novo formation of branches along the axon shaft and their directed growth into targets. We will study the roles of BMP-3 and a novel secreted CAM-like protein with 6 Ig domains and a MAM domain (temporarily named "Ig6M"), which were isolated using a differential display PCR screen to identify candidate target recognition molecules for corticospinal axons. We will use in vitro axon guidance and branching assays, as well as gain-of-function and loss-of-function genetic experiments in mice, to determine the sufficiency and requirement of BMP-3 and Ig6M in controlling the formation and directed growth of corticospinal axon branches.